Underutilization of the V kappa 10C gene in the B cell repertoire is due to the loss of productive VJ rearrangements during B cell development.

The V kappa10 family of murine light chain Ig genes is composed of three members, two of which (V kappa 10A and V kappa 10B) are well used. V kappa 10C, the third member of this family, is not detected in any expressed Abs. Our previous work showed that V kappa 10C is structurally functional and can recombine, but mRNA levels in spleen were extremely low relative to those of V kappa 10A and V kappa 10B. Furthermore, while the V kappa 10C promoter was efficient in B cells, it was shown to work inefficiently in pre-B cell lines. Here, we extend our analysis of the V kappa 10 family and examine V kappa 10 gene accessibility, their representation in V kappa cDNA phage libraries, and the frequency and nature of rearrangements during different stages of B cell development. We demonstrate that V kappa 10C is under-represented in V kappa cDNA libraries, but that the frequency of its sterile transcripts in pre-B cells surpasses both V kappa 10A and V kappa 10B, indicating that the gene is as accessible as V kappa 10A and V kappa 10B to the recombination machinery. We also demonstrate that V kappa 10C recombines at a frequency equal to that of V kappa 10A in pre-B cells and has a normal nonproductive to productive recombination ratio. As B cells develop, however, both the frequency of V kappa 10C rearrangements and the presence of productive rearrangements decline, indicating that these cells are in some fashion being eliminated.

Coreceptor competition for association with CD4 may change the susceptibility of human cells to infection with T-tropic and macrophagetropic isolates of human immunodeficiency virus type 1.

The chemokine receptors CCR5 and CXCR4 were found to function in vivo as the principal coreceptors for M-tropic and T-tropic human immunodeficiency virus (HIV) strains, respectively. Since many primary cells express multiple chemokine receptors, it was important to determine if the efficiency of virus-cell fusion is influenced not only by the presence of the appropriate coreceptor (CXCR4 or CCR5) but also by the levels of other coreceptors expressed by the same target cells. We found that in cells with low to medium surface CD4 density, coexpression of CCR5 and CXCR4 resulted in a significant reduction in the fusion with CXCR4 domain (X4) envelope-expressing cells and in their susceptibility to infection with X4 viruses. The inhibition could be reversed either by increasing the density of surface CD4 or by antibodies against the N terminus and second extracellular domains of CCR5. In addition, treatment of macrophages with a combination of anti-CCR5 antibodies or beta-chemokines increased their fusion with X4 envelope-expressing cells. Conversely, overexpression of CXCR4 compared with CCR5 inhibited CCR5-dependent HIV-dependent fusion in 3T3.CD4.401 cells. Thus, coreceptor competition for association with CD4 may occur in vivo and is likely to have important implications for the course of HIV type 1 infection, as well as for the outcome of coreceptor-targeted therapies.

Roles for insulin receptor, PI3-kinase, and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells.

BACKGROUND: Previously, we demonstrated that insulin stimulates production of nitric oxide (NO) in endothelial cells. However, specific insulin-signaling pathways mediating production of NO have not been elucidated. METHODS AND RESULTS: We developed methods for transfection of human umbilical vein endothelial cells (HUVECs) and direct measurement of NO to begin defining insulin-signaling pathways related to NO production. HUVECs were cotransfected with enhanced Green Fluorescent Protein (eGFP) and another gene of interest. Transfection efficiencies >95% were obtained by selecting cells expressing eGFP. Overexpression of insulin receptors in HUVECs resulted in an approximately 3-fold increase in production of NO in response to insulin. In contrast, HUVECs overexpressing a tyrosine kinase-deficient mutant insulin receptor had a dose-response curve similar to that of control cells. Overexpression of inhibitory mutants of either phosphatidylinositol 3-kinase (PI3K) or Akt resulted in nearly complete inhibition of insulin-stimulated production of NO. Overexpression of an inhibitory mutant of Ras had a much smaller effect. CONCLUSIONS: Receptor kinase activity is necessary to mediate production of NO through the insulin receptor. Both PI3K and Akt contribute importantly to this process, whereas the contribution of Ras is small.

Differential effects of Cbl and 70Z/3 Cbl on T cell receptor-induced phospholipase Cgamma-1 activity.

We demonstrate that the differential effects Cbl and oncogenic 70Z/3 Cbl have on Ca(2+)/Ras-sensitive NF-AT reporters is partially due to their opposing ability to regulate phospholipase Cgamma1 (PLCgamma1) activation as demonstrated by analysis of the activation of an NF-AT reporter construct and PLCgamma1-mediated inositol phospholipid (PI) hydrolysis. Cbl over-expression resulted in reduced T cell receptor-induced PI hydrolysis, in the absence of any effect on PLCgamma1 tyrosine phosphorylation. In contrast, expression of 70Z/3 Cbl led to an increase in basal and OKT3-induced PLCgamma1 phosphorylation and PI hydrolysis. These data indicate that Cbl and 70Z/3 Cbl differentially regulate PLCgamma1 phosphorylation and activation. The implications of these data on the mechanism of Cbl-mediated signaling regulation are discussed.

Vaccination with DNA encoding internal proteins of influenza virus does not require CD8(+) cytotoxic T lymphocytes: either CD4(+) or CD8(+) T cells can promote survival and recovery after challenge.

DNA vaccination offers the advantages of viral gene expression within host cells without the risks of infectious virus. Like viral vaccines, DNA vaccines encoding internal influenza virus proteins can induce immunity to conserved epitopes and so may defend the host against a broad range of viral variants. CD8(+) cytotoxic T lymphocytes (CTL) have been described as essential effectors in protection by influenza nucleoprotein (NP), although a lesser role of CD4(+) cells has been reported. We immunized mice with plasmids encoding influenza virus NP and matrix (M). NP + M DNA allowed B6 mice to survive otherwise lethal challenge infection, but did not protect B6-beta(2)m(-/-) mice defective in CD8(+) CTL. However, this does not prove CTL are required, because beta(2)m(-/-) mice have multiple immune abnormalities. We used acute T cell depletion in vivo to identify effectors critical for defense against challenge infection. Since lung lymphocytes are relevant to virus clearance, surface phenotypes and cytolytic activity of lung lymphocytes were analyzed in depleted animals, along with lethal challenge studies. Depletion of either CD4(+) or CD8(+) T cells in NP + M DNA-immunized BALB/c mice during the challenge period did not significantly decrease survival, while simultaneous depletion of CD4(+) and CD8(+) cells or depletion of all CD90(+) cells completely abrogated survival. We conclude that T cell immunity induced by NP + M DNA vaccination is responsible for immune defense, but CD8(+) T cells are not essential in the active response to this vaccination. Either CD4(+) or CD8(+) T cells can promote survival and recovery in the absence of the other subset.

Human NK cells express endothelial nitric oxide synthase, and nitric oxide protects them from activation-induced cell death by regulating expression of TNF-alpha.

Although NO appears important in rodent immune responses, its involvement in the human immune system is unclear. We report that human NK cells express constitutive endothelial NO synthase mRNA and protein, but not detectable levels of inducible NO synthase. They produce NO following activation by coculture with target cells or cross-linking with anti-CD16 mAb, and production is increased in the presence of IL-2. N-monomethyl-L-arginine (L-NMA), a NOS inhibitor, partially inhibited NK cell lysis of four different target cells (<40% inhibition at 500 microM L-NMA), but not granule release following coculture with target cells, or Fas ligand induction following cross-linking with anti-CD16 mAb. However, L-NMA augmented apoptosis of NK cells induced by activation through CD16 ligation or coculture with K562. An NO donor, S-nitroso-N-acetylpenicillamine (SNAP), suppressed apoptosis of NK cells induced by CD16 cross-linking or coculture with target cells, suggesting that endogenous NO production is involved in protection of NK cells from activation-induced apoptosis, thereby maintaining NK activity. SNAP also suppressed, and L-NMA enhanced, expression of TNF-alpha, reported to be involved in activation-induced NK cell death, in response to CD16 cross-linking. Suppression of anti-CD16-induced apoptosis by SNAP was reversed by the addition of rTNF-alpha. DNA-binding activity of the transcription factor, NF-AT, which is involved in TNF-alpha induction upon ligation of CD16, was inhibited by SNAP and enhanced by L-NMA. Our results suggest that down-regulation of TNF-alpha expression, possibly due to suppression of NF-AT activation, is a mechanism by which endogenous NO protects NK cells from activation-induced apoptosis, and maintains lytic capacity.

Bax is frequently compromised in Burkitt's lymphomas with irreversible resistance to Fas-induced apoptosis.

We have analyzed the Fas-mediated death pathway in a panel of 11 Epstein-Barr virus (EBV)-negative and 10 EBV-positive Burkitt's lymphoma (BL) cell lines. We show that the increased expression of Fas in EBV-positive cell lines is mediated via LMP-1. Four of the 21 BL cell lines are readily responsive to Fas-mediated cell death signals. Of the remaining 17 cell lines, 10 can be sensitized by up-regulating Fas either via exogenous expression of LMP-1 or via treatment with CD40L. These same cell lines can also be sensitized by treatment with cycloheximide (CHX), which, however, does not result in up-regulation of Fas. Neither up-regulation of Fas, nor treatment with CHX, restore Fas sensitivity in seven BL cell lines. Further analyses indicated that 5 of the 7 cell lines (and none of the 14 responsive cell lines) were also compromised in the integrity/expression of the proapoptotic gene Bax. Thus, in most BL cell lines, the Fas pathway seems to be inhibited, although the mechanism of inhibition varies. The correlation between Bax mutation and irreversible (by CD40L or CHX) Fas resistance raises the possibility, for the first time, that Bax may play a critical function in Fas-mediated cell death in BL.

Fas ligand induction in human NK cells is regulated by redox through a calcineurin-nuclear factors of activated T cell-dependent pathway.

Fas ligand (FasL) on cytotoxic lymphocytes is important for mediating apoptosis of activated lymphocytes and other target cells. We have reported that NK cell functions, such as proliferation, cell death, and killing activity, are subject to regulation by cellular redox status. Here, we report that expression of FasL protein and mRNA in activated NK cells is also regulated by redox. Ligation of CD16 on IL-2-preactivated NK cells resulted in reduction of intracellular peroxide level as well as induction of FasL expression. This CD16-induced FasL expression was suppressed by oxidative stress, including thiol deprivation or treatment with hydrogen peroxide (H2O2). Addition of thiol-reducing compounds, such as L-cystine, 2-ME, or N-acetyl cysteine, restored FasL expression. These data suggest that CD16 stimulation requires cellular reducing status for FasL induction in NK cells. Because FasL gene activation following CD16 cross-linking is regulated by the NF of activated T cells (NFAT), we examined the effect of oxidative stresses on NFAT activation. Electrophoretic mobility shift assays revealed that both thiol insufficiency and H2O2 treatment suppressed DNA-binding activity of NFAT and that addition of thiol-reducing compounds reversed or even enhanced it. Furthermore, these oxidative stresses inhibited activity of calcineurin, a serine/threonine phosphatase that regulates NFAT activation. These results suggest that suppression of calcineurin and NFAT activation is a mechanism by which oxidative stress inhibits FasL induction in activated NK cells and further support the hypothesis that thiol-reducing compounds might be required for maintenance of optimal NK functions under physiologic oxidative conditions.

Cytokines regulate expression and function of the HIV coreceptor CXCR4 on human mature dendritic cells.

HIV-infected dendritic cells (DC) efficiently transmit infection to CD4+ T cells during the process of T cell activation. To further understand interactions between DC and HIV, cytokine regulation of HIV coreceptors on cultured Langerhans cells (cLC, as prototypes of mature DC) was studied. Expression of cell surface CXCR4 on cLC was up-regulated by IL-4 and TGF-beta1 and inhibited by IFN-alpha, IFN-beta, and IFN-gamma, whereas cytokines did not appreciably regulate CCR5. Changes in cell surface CXCR4 expression on cLC correlated with T cell-tropic (X4)-HIV envelope-mediated syncytium formation and X4-HIV infection levels. A relative increase in the ratio of type 2/type 1 cytokine production, which can occur in HIV disease, may up-regulate CXCR4 expression on mature DC and promote infection by X4 viruses. Importantly, these findings suggest that cytokine dysregulation may be linked to the emergence of X4-HIV strains as HIV-infected individuals progress to AIDS.

Production of IL-10 by human natural killer cells stimulated with IL-2 and/or IL-12.

Human NK cell activity can be augmented in vitro by stimulation with IL-2 or IL-12, both of which also induce the production of IFN-gamma, TNF-alpha, and granulocyte-macrophage CSF by NK cells. For the first time, we demonstrate that freshly purified NK cells stimulated with IL-2 proliferated and produced IL-10 in a dose-dependent manner. IL-10 mRNA expression, as detected by semiquantitative reverse transcription-PCR, reached peak levels at 24 h. IL-10 protein was detectable on day 2 and further increased on days 3 and 6 as measured by ELISA. However, IL-12 alone induced neither substantial proliferation nor detectable IL-10 production by fresh NK cells, but it synergized with IL-2 in inducing IL-10 mRNA expression and protein synthesis. IL-10 production by activated NK cells was confirmed by intracytoplasmic cytokine staining by three-color immunofluorescence of CD16+ and/or CD56+ NK cells with anti-IL-10 antibody. IL-10 production by NK cells was further confirmed in the NK-like cell line, YT, which constitutively expressed IL-10 mRNA and protein. IL-12 alone did not induce NK proliferation, but it inhibited IL-2-induced proliferation. Neutralization of endogenously produced IL-10 with anti-IL-10 antibodies did not overcome the inhibition of IL-2-induced proliferation by IL-12. Together, these results demonstrate that IL-2 and IL-12 synergize to induce IL-10 production by human NK cells and that IL-12 inhibits IL-2 induced NK cell proliferation by an IL-10-independent mechanism.

Expression and function of CCR5 and CXCR4 on human Langerhans cells and macrophages: implications for HIV primary infection.

Transmission of HIV-1 is predominantly restricted to macrophage (Mphi)-tropic strains. Langerhans cells (LCs) in mucosal epithelium, as well as macrophages located in the submucosal tissues, may be initial targets for HIV-1. This study was designed to determine whether restricted transmission of HIV-1 correlates with expression and function of HIV-1 co-receptors on LCs and macrophages. Using polyclonal rabbit IgGs specific for the HIV co-receptors cytokines CXCR4 and CCR5, we found that freshly isolated epidermal LCs (resembling resident mucosal LCs) expressed CCR5, but not CXCR, on their surfaces. In concordance with surface expression, fresh LCs fused with Mphi-tropic but not with T-tropic HIV-1 envelopes. However, fresh LCs did contain intracellular CXCR4 protein that was transported to the surface during in vitro culture. Macrophages expressed high levels of both co-receptors on their surfaces, but only CCR5 was functional in a fusion assay. These data provide several possible explanations for the selective transmission of Mphi-tropic HIV variants and for the resistance to infection conferred by the CCR5 deletion.

Role of the p53 tumor suppressor gene in the tumorigenicity of Burkitt's lymphoma cells.

Burkitt's lymphoma (BL) cell lines carry a translocated c-myc gene and, in 60-80% of cases, exhibit mutations in the p53 tumor suppressor gene. We examined the potential role of the p53 gene in BL tumorigenicity using an in vitro assay that measures p53-dependent cell cycle arrest in the G1 phase of the cell cycle and an in vivo athymic murine model that detects differences in the tumorigenicity of BL cell lines. A highly significant inverse correlation was found between the ability of BL cells to arrest in G1 after irradiation and their tumorigenicity in athymic mice, consistent with the notion that loss of p53 function is associated with increased tumorigenicity. Inactivation of wild-type (wt) p53 function by expression of the human papillomavirus E6 protein in the AG876V BL cell line, which carries both wt and mutant p53 proteins, rendered the cell line significantly more tumorigenic in athymic mice. Transfection of the wt p53 gene into the p53 mutant and highly tumorigenic BL-41 cell line caused it to acquire wt p53 function and rendered it less tumorigenic in mice. In addition to confirming a role for the loss of p53 function in tumor progression, the data demonstrate that wt p53 protein can reduce BL tumorigenicity in vivo.

Target cell-induced apoptosis of interleukin-2-activated human natural killer cells: roles of cell surface molecules and intracellular events.

We previously reported that natural killer (NK)-sensitive target cells, K562, kill interleukin-2-stimulated (lymphokine-activated killer [LAK]) but not unstimulated NK cells. We have now investigated the molecular basis of this phenomenon. Soluble monoclonal antibody (MoAb) to CD18 inhibited 75% of K562-induced DNA fragmentation and membrane disruption, whereas blocking MoAb to Fas partially inhibited only the DNA fragmentation. MoAbs to CD2, CD11a, CD11b, B7, or CD16 had limited or no effect on K562-induced death of LAK cells. Receptor ligation with either immobilized MoAb to CD18 or Fas induced membrane disruption and DNA degradation in LAK cells independently of K562, and MoAb to CD18, CD11a, or CD11b enhanced DNA fragmentation induced by anti-Fas. Fas-L-transfected Raji cells also killed LAK cells, but only if Fas-L expression was amplified. K562 cells rapidly triggered protein phosphorylation in LAK cells, and the tyrosine kinase inhibitor, Herbimycin A, inhibited DNA fragmentation and membrane disruption. Protease inhibitors strongly suppressed K562-mediated DNA fragmentation of LAK cells, but not membrane disruption. In conclusion, (1) K562-induced death of LAK cells involves primarily CD18, although other molecules, such as Fas, may also be involved; (2) K562-mediated apoptosis of LAK cells requires tyrosine phosphorylation and protease activity; (3) engagement of Fas by immobilized MoAb or Fas-L on target cells can also kill LAK cells; and (4) Fas-immobilized MoAb synergizes with coimmobilized MoAb to CD11a, CD11b, or CD18 for LAK cell killing. Activation-induced death of NK cells may represent a mechanism for NK cell regulation.

IL-12 administered in vivo to young and aged mice. Discrepancy between the effects on tumor growth in vivo and cytotoxic T lymphocyte generation ex vivo: dependence on IFN-gamma.

Because IL-12 restores allogeneic cytotoxic T lymphocyte (CTL) activity by T cells of aged mice in vitro, we initially assessed whether IL-12 could overcome age-related deficits when given to aged mice in vivo. Growth of P815(H-2(d)) was enhanced in aged compared with young BALB/c (H-2(d)) mice and tumor growth was curtailed by IL-12 in both age groups. Unexpectedly, secondary CTL stimulated ex vivo with P815 were reduced in IL-12-treated mice compared with controls. Primary CTL generated ex vivo across MHC differences in IL-12 treated BALB/c and C57BL/6 young mice were reduced by 90-99%, were dose- and time-dependent, and were associated with reduced allo-stimulated NK-like activity and [3H]thymidine incorporation. IFN-gamma was elevated in sera and in supernatants from allo-stimulated cultures from IL-12-treated mice, while IL-4 was reduced in such supernatants, suggesting that, despite reduced CTL, IL-12 was associated with increased Th1- and reduced Th2-type cytokine production. IL-12 also induced splenomegaly, primarily due to increased numbers of cells lacking markers of mature T, B and NK cells, or macrophages, or polymorphonuclear leukocyte morphology. IFN-gamma mutant mice exhibited reduced splenic enlargement in response to IL-12, suggesting that the splenomegaly was due, in part, to IFN-gamma production. However, reduced CTL generation was not due entirely to dilution of CTL precursor cells because spleen cellularity and size increased 3-fold while CTL activity decreased 10- to 100-fold, and CTL generation normalized to CD8(+) T effector cells was still significantly reduced in IL-12-treated mice. Interestingly, purified CD4(+) and CD8(+) T cells from IL-12-treated normal mice exhibited greater proliferative and cytolytic activities respectively compared with controls. Thus, effector T cells in IL-12-treated mice were not impaired, but exhibited augmented responsiveness, suggesting that IL-12 induced complex interactions among spleen cell populations and that these effects, in part, are mediated by IFN-gamma.

CD4-IgG binding threshold for inactivation of human immunodeficiency virus type 1.

The stoichiometry of human immunodeficiency virus type 1 (HIV-1) inactivation by soluble receptor CD4-IgG hybrid dimers (CD4-IgG) was examined. The extent of HIV-1 inactivation was measured in a sensitive plaque-forming assay, and the corresponding level of CD4-IgG binding was determined by immunofluorescence of infected cells. Ninety percent virus inactivation occurred at relatively low levels of CD4-IgG binding (10% of the saturating level). At even lower binding levels (1.4% of maximum binding), virus survival was 44%. Over a broad range of binding conditions, the survival curve followed a model in which viruses binding more than a threshold level of CD4-IgG were completely inactivated, while viruses binding less remained infectious. The data indicate that CD4-IgG binding to 1.4% of gp120 binding sites equals the threshold for inactivation. Thus, virus inactivation can begin when 3 CD4-IgG (of approximately 216 gp120 sites) bind per virion.

Mitogenic stimulation of human lymphocytes mediated by a cell surface elastase.

A ca. 45-kDa protein which was recently identified and purified to homogeneity from a solid tumor cell line as a T-cell mitogen was found to have significant sequence similarities with human monocyte/neutrophil elastase inhibitor (EI) [1]. Since EI is a known substrate for elastase, a determination of whether a cell surface expressed elastase-like molecule might be the binding protein for this 45-kDa factor and mediate mitogenic signal transduction was undertaken. First, the surface of tumor infiltrating lymphocytes, TIL 660, the indicator cell line used for the purification of this mitogen, was shown to stain positively with an anti-elastase antibody using flow cytofluorometry for quantitation. Then, after observing an inverse correlation between cell surface staining and the proliferative status of the TILs, behavior which might be expected of a growth factor receptor upon activation, mitogenic signal transduction was attempted through the elastase-like molecules of the lymphocytes' plasma membrane with the anti-elastase antibody in the role of mitogen. A greater than 4-fold mitogenic stimulation was observed when this antibody was covalently linked to latex beads; in contrast, addition of the soluble form of the same antibody did not result in any increase in [3H]thymidine incorporation into the cells' DNA. Hence, these data support induced clustering of an elastase-like molecule on the lymphocyte surface as a mediator of mitogenesis and suggest that the binding protein for mitogenic signal transduction induced by the 45-kDa protein, a member of the serine protease inhibitor (serpin) superfamily of proteins, is a molecule with structure similar to a serine protease.

Expression of a constitutive mutant of iron regulatory protein 1 abolishes iron homeostasis in mammalian cells.

Iron regulatory proteins (IRPs) are iron-sensing proteins that bind to RNA stem-loop sequences known as iron-responsive elements (IREs) when cells are depleted of iron. Although IRPs have been shown to bind to IREs derived from ferritin and transferrin receptor (TfR) mRNAs in vitro, there has not been a direct demonstration of the impact of a recombinant IRP on the expression of endogenous IRE-containing transcripts. In this study, we evaluate the impact of expression of C437S, a mutant of IRP1 that binds IREs regardless of cellular iron status, on the regulation of biosynthesis of ferritin and TfR. Despite being made iron-replete, cells expressing C437S continue to synthesize and express high amounts of TfR, while the synthesis of ferritin is repressed. Thus, a single mutant IRP can prevent the usual homeostatic changes in ferritin and TfR biosynthesis. Cells expressing the mutant protein would therefore be predicted to be unable to defend against iron overload. Preliminary results show that cells treated with iron have diminished cell survival when C437S is expressed, and we have thus created a tissue culture model system for the study of iron toxicity.

Mediation of human immunodeficiency virus type 1 binding by interaction of cell surface heparan sulfate proteoglycans with the V3 region of envelope gp120-gp41.

The mechanism of heparan sulfate (HS)-mediated human immunodeficiency virus type 1 (HIV-1) binding to and infection of T cells was investigated with a clone (H9h) of the T-cell line H9 selected on the basis of its high level of cell surface CD4 expression. Semiquantitative PCR analysis revealed that enzymatic removal of cell surface HS by heparitinase resulted in a reduction of the amount of HIV-1 DNA present in H9h cells 4 h after exposure to virus. Assays of the binding of recombinant envelope proteins to H9h cells demonstrated a structural requirement for an oligomeric form of gp120/gp41 for HS-dependent binding to the cell surface. The ability of the HIV-1 envelope to bind simultaneously to HS and CD4 was shown by immunoprecipitation of HS with either antienvelope or anti-CD4 antibodies from 35SO4(2-)-labeled H9h cells that had been incubated with soluble gp140. Soluble HS blocked the binding of monoclonal antibodies that recognize the V3 and C4 domains of the envelope protein to the surface of H9 cells chronically infected with HIV-1IIIB. The V3 domain was shown to be the major site of envelope-HS interaction by examining the effects of both antienvelope monoclonal antibodies and heparitinase on the binding of soluble gp140 to H9h cells.

Does the age-related change in CD44-defined T-cell subsets have functional significance for cytotoxic T lymphocyte generation?

CD44 or Pgp-1 is a transmembrane leukocyte adhesion-related glycoprotein which is often expressed in greater density on the membranes of memory T lymphocytes (CD44hi) compared to naive T cells (CD44lo). The proportion of Pgphi or CD44hi cells among T cells is increased with advancing age. We examined the relevance of this alteration for the age-related decrease in the generation of allospecific CTL activity. The findings confirm the age-related increase in the frequency of CD44hi cells in spleens of aged mice of several strains, but also show interstrain variability in the magnitude of the increase (bm1 > C57BL/6 > BALB/c). In contrast, we found that after allo-stimulation, the proportion of cells bearing the memory phenotype is decreased in cells from aged mice, particularly within the CD8+ T cell subset. To determine if these observations reflected an alteration in the frequency or responsiveness of naive T cells, enriched populations of spleen cells depleted of CD44hi cells were prepared from spleen cells of young and aged mice, and stimulated in mixed lymphocyte culture. Enrichment for cells expressing the naive phenotype did not restore the ability of T cells from aged mice to generate allospecific CTL. Together, these findings suggest that (1) the age-related increase in frequency of splenic T cells expressing memory phenotype and concordant decrease in phenotypically naive cells, does not explain the age-related decrease in the ability to generate primary allo-CTL, and (2) naive cells from aged mice exhibit intrinsically compromised ability to generate CTL in response to primary alloantigenic stimulation.

Effects of IL-12 on the generation of cytotoxic activity in human CD8+ T lymphocytes.

We have studied the effects of human rIL-12 on the proliferation and generation of cytotoxic activity in human CTL precursors. Purified human blood CD8+ T lymphocytes were stimulated overnight with immobilized alpha-CD3 and cultured 3 to 4 additional days under various conditions. The addition of IL-12 resulted in a marked (10- to 20-fold), dose-dependent, augmentation of cytotoxicity per cell with a smaller (2-fold) increase in cell number. IL-12 augmentation of proliferation and cytotoxicity of CD8+ T cells was not inhibited by a mAb to the p55 subunit of the IL-2 receptor (alpha-Tac) at a concentration sufficient to block the activity of exogenously added IL-2, indicating that the activity of IL-12 did not require IL-2. Addition of IL-12 at the time of alpha-CD3 activation or 1 day later was highly effective at augmenting cytotoxicity, whereas delayed addition of IL-12 (day 2 or 3) resulted in a smaller increase in CTL activity with no increase in cell number. IL-12 at all doses tested synergized with low dose IL-2 in inducing the proliferation and differentiation of CD8+ T cells. The synergistic effect was not blocked by adding neutralizing serum to IFN-gamma. In contrast to this synergistic effect, IL-12 significantly inhibited the proliferation observed in the presence of higher concentrations of IL-2 (4,500 and 13,500 pg/ml). An inhibitory effect of IL-12 was also observed when IL-12 was added to CD8+ T lymphocytes 3 days subsequent to activation with alpha-CD3 and IL-2. This broad set of potent effects of IL-12 on CD8+ T cell responses suggests that IL-12 may play an important immunoregulatory role on CTL development in vivo and may be a useful tool for manipulating this process in vivo for investigational and immunotherapeutic purposes.